Genetic locus imparting a low anatabine trait in tobacco and methods of using

ABSTRACT

Provided herein are genetic markers and a coding sequence associated with a low- or ultra-low anatabine trait in tobacco.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCELISTING

This application is a continuation of U.S. patent application Ser. No.14/794,092, filed Jul. 8, 2015, which claims the benefit of U.S.Provisional Application No. 62/021,738, filed Jul. 8, 2014, both ofwhich are incorporated by reference in their entireties herein. Asequence listing contained in the file named “P34624US02_SL.TXT” whichis 21,951 bytes (measured in MS-Windows®) and created on Mar. 20, 2020,is filed electronically herewith and incorporated by reference in itsentirety.

TECHNICAL FIELD

This disclosure relates to genetic markers and the coding sequenceassociated with the locus that imparts a low anatabine trait in tobacco.

BACKGROUND

Anatabine is a minor alkaloid in tobacco and it is also a precursor forN′-nitrosoanatabine (NAT), one of four tobacco-specific nitrosamines(TSNAs) found in cured tobacco. Even though anatabine constitutes onlyabout 3% of the total alkaloids found in tobacco, NAT constitutes about40%-50% of the total amount of TSNAs. Therefore, reducing the amount ofanatabine in a tobacco plant may be a viable way to reduce the overallamount of TSNAs in cured tobacco.

SUMMARY

This disclosure describes a correlation between a low anatabine traitand the quinolinate synthase (QS) gene. The correlation is useful forunderstanding the alkaloid synthesis pathway and its regulation,particularly with respect to anatabine. Single nucleotide polymorphisms(SNPs) in the QS gene also provide markers for breeding the lowanatabine or ultra-low anatabine (ULA) trait into tobacco lines.

In one aspect, a tobacco hybrid, variety, line, or cultivar is providedthat includes plants having a mutation in an endogenous nucleic acidhaving the sequence shown in SEQ ID NO: 7. In some embodiments, themutant plants exhibit reduced expression or activity of QS. In someembodiments, leaf from the mutant plants exhibit reduced amounts of atleast one alkaloid (e.g., anatabine). In some embodiments, cured leaffrom the mutant plants exhibit a reduced amount of at least one TSNA(e.g., N′-nitrosoanatabine (NAT)).

In another aspect, seed produced by any of the tobacco hybrids,varieties, lines, or cultivars described herein, the seed including themutation in an endogenous nucleic acid having the sequence shown in SEQID NO: 7.

In still another aspect, a method of making a tobacco plant is provided.Such a method typically includes the steps of inducing mutagenesis inNicotiana tabacum cells to produce mutagenized cells; obtaining one ormore plants from the mutagenized cells; and identifying at least one ofthe plants that comprises a mutation in an endogenous nucleic acidhaving the sequence shown in SEQ ID NO: 7.

In some embodiments, such a method further includes identifying at leastone of the plants that comprises leaf exhibiting a reduced amount of atleast one alkaloid relative to leaf from a plant lacking the mutation.In some embodiments, the at least one alkaloid is anatabine. In someembodiments, the method further includes identifying at least one of theplants that comprises leaf that, when cured, exhibit a reduced amount ofat least one TSNA relative to cured leaf from a plant lacking themutation. In some embodiments, the at least one TSNA is NAT.

In some embodiments, mutagenesis is induced using a chemical mutagen orionizing radiation. Representative chemical mutagens include, withoutlimitation, nitrous acid, sodium azide, acridine orange, ethidiumbromide, and ethyl methane sulfonate (EMS). Representative ionizingradiation includes, without limitation, x-rays, gamma rays, fast neutronirradiation, and UV irradiation. In some embodiments, mutagenesis isinduced using TALEN. In some embodiments, mutagenesis is induced usingzinc-finger technology.

In yet another aspect, a method for producing a tobacco plant isprovided. Such a method typically includes the steps of: crossing atleast one plant of a first tobacco line with at least one plant of asecond tobacco line, the plant of the first tobacco line having amutation in an endogenous nucleic acid having the sequence shown in SEQID NO: 7; and selecting for progeny tobacco plants that have themutation.

In some embodiments, the method further includes selecting for progenytobacco plants that comprise leaf exhibiting reduced expression oractivity of QS relative to leaf from a plant lacking the mutation. Insome embodiments, the method further includes selecting for progenytobacco plants that comprise leaf exhibiting a reduced amount of atleast one alkaloid relative to leaf from a plant lacking the mutation.In some embodiments, the at least one alkaloid is anatabine. In someembodiments, the method further includes selecting for progeny tobaccoplants that comprise leaf that, when cured, exhibit a reduced amount ofat least one TSNA relative to cured leaf from a plant lacking themutation. In some embodiments, the at least one TSNA is NAT.

In still another aspect, a tobacco product is provided that includescured leaf from a tobacco plant having a mutation in an endogenousnucleic acid having the sequence shown in SEQ ID NO: 7. In someembodiments, the leaf exhibits reduced expression or activity of QSrelative to leaf from a plant lacking the mutation. In some embodiments,the leaf exhibits a reduced amount of at least one alkaloid relative toleaf from a plant lacking the mutation. A representative alkaloid isanatabine. In some embodiments, the cured leaf exhibits a reduced amountof at least one TSNA relative to cured leaf from a plant lacking themutation. A representative TSNA is NAT.

In one aspect, a method of producing a tobacco product is provided. Sucha method typically includes providing cured leaf from a tobacco planthaving a mutation in an endogenous nucleic acid having the sequenceshown in SEQ ID NO: 7; and manufacturing a tobacco product using thecured leaves. In some embodiments, the cured leaf exhibits reducedexpression or activity of QS relative to cured leaf from a plant lackingthe mutation. In some embodiments, the cured leaf exhibits a reducedamount of at least one alkaloid relative to cured leaf from a plantlacking the mutation. A representative alkaloid is anatabine. In someembodiments, the cured leaf exhibits a reduced amount of at least oneTSNA relative to cured leaf from a plant lacking the mutation. Arepresentative TSNA is NAT.

Any of the mutations described herein can include, without limitation, apoint mutation, an insertion, a deletion, and a substitution.

In another aspect, a method of screening plants is provided. Such amethod typically includes providing plant material from a plant asdescribed herein; and determining the level of expression or activity ofQS in the plant material. In still another aspect, a method of screeningplants is provided. Such a method typically includes providing plantmaterial from a plant as described herein; and determining the amount ofat least one alkaloid in the plant material. In some embodiments, the atleast one alkaloid is anatabine. In yet another aspect, a method ofscreening plants is provided. Such a method typically includes providingplant material from a plant as described herein; curing the plantmaterial; and determining the amount of at least one TSNA in the plantmaterial. In some embodiments, the at least one TSNA is NAT. In any ofthe methods described herein, the plant tissue can be leaf.

In another aspect, a method of identifying a tobacco plant or tobaccogermplasm having a low or ultra-low anatabine phenotype is provided.Such a method typically includes detecting, in a tobacco plant ortobacco germplasm, at least one simple sequence repeat (SSR) of a markerlocus that is associated with the phenotype, wherein the marker locus isrepresented by at least one of SEQ ID NOs: 1-6 or displays arecombination frequency of less than 10% with respect to a marker locusrepresented by at least one of SEQ ID NOs: 1-6 and maps to Linkage Group6. In some embodiments, the detecting includes amplifying the markerlocus or a portion of the marker locus with a primer pair and detectingthe resulting amplicon. Representative primer pairs include, withoutlimitation, SEQ ID NOs: 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17and 18, and 19 and 20.

In another aspect, a method of breeding is provided that includescrossing the tobacco plant or germplasm identified by a method describedherein with a second tobacco plant or germplasm. In some embodiments,such a method further includes one or more steps of backcrossing,selfing, outcrossing, and selection of progeny plants. In someembodiments, such a method further includes the step of performingmolecular marker analysis on DNA samples isolated from one or moreprogeny plants, wherein said analysis identifies a plant that includesat least one SSR selected from the group consisting of SEQ ID NOs: 1-6,wherein the SSR is associated with reduced amounts of anatabine in theprogeny plants. In some embodiments, such a method further includesdetermining the amount of anatabine in leaf of the identified plant.

In yet another aspect, a method for detecting the presence of a markerlinked to a gene associated with low or ultra-low anatabine in a tobaccoplant is provided. Such a method typically includes analyzing chromosome6 of the tobacco plant and detecting the presence of at least one SSRmarker having a sequence selected from the group consisting of SEQ IDNOs: 1-5 and 6, and linked to a gene associated with low or ultra-lowanatabine, wherein the presence of the at least one marker is indicativeof a plant that exhibits low or ultra-low amounts of anatabine.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the anatabine profiles of F2 individualplants.

FIG. 2 depicts the results of experiments performed to examine theinheritance pattern of anatabine. Panel A shows the anatabineinheritance profile and Panel B is a graph showing the averagepercentage of anatabine for an F1 cross between the FC401 mutant #1(MS4144)×FC401 Mutant #2 (MS4360).

FIG. 3 is a graph showing the amount of anatabine in the root and leafof reference variety tobacco plants and the two mutant tobacco plants.

FIG. 4 is an alignment of a portion of the QS sequence containing themutations (SEQ ID NOs: 45-56 (top to bottom)).

FIG. 5 is an alignment of QS sequences (SEQ ID NOs: 34-44 (top tobottom)).

FIG. 6 is an image showing co-segregation of the mutants with anatabine.

DETAILED DESCRIPTION

Currently, there are no commercial tobacco varieties that exhibitreduced amounts of anatabine, and, prior to this disclosure, there wereno known markers or genes associated with an ultra-low anatabine traitor even a low anatabine trait. As used herein, tobacco plants carryingan “ultra-low anatabine” trait (i.e., “ultra-low anatabine plants”) arethose plants having about 10% or less (e.g., about 9%, about 8%, about7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1% or less(e.g., about 0%)) of the amount of anatabine that is typically presentin a reference variety as shown in Table 1.

TABLE 1 Reference Varieties Types Reference Varieties Burley TN 90 LC,KT 204 LC, TN 86 LC, KDH959 Dark KY171, NL Madole, VA359, TR Madole FlueK326, K346, K394 Oriental Izmir, Basma Drama, Basma Zihna I, MarylandMaryland 609, Maryland 60, Maryland TI228 Cigar Caujaro, Florida 2612,Galpao

A skilled artisan would appreciate that this corresponds to a reductionin the amount of anatabine, relative to a reference variety tobaccoplant, by at least about 90% (e.g., at least about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% reduction)). As used herein, tobacco plants carrying a “lowanatabine” trait (i.e., “low anatabine plants”) are those plants havingbetween about 50% and about 89% (e.g., between about 55% and about 85%,between about 60% and about 80%, between about 65% and about 75%,between about 50% and about 75%, between about 50% and about 60%,between about 60% and about 70%, or between about 70% and about 89%) ofthe amount of anatabine that is typically present in a reference varietytobacco plant. A skilled artisan would appreciate that this correspondsto a reduction in the amount of anatabine, relative to a referencevariety tobacco plant, of between about 11% and about 50% (e.g., betweenabout 15% and about 45%, between about 20% and about 40%, between about25% and about 35%, between about 15% and about 30%, between about 25%and about 45%, or between about 30% and about 50%).

As described herein, six simple sequence repeat (SSR) markers wereidentified that are tightly linked with the low anatabine trait inNicotiana tabacum. The sequence of each of these markers is shown in SEQID NOs: 1-6. These markers can be used for breeding the low anatabinetrait into varieties of interest. The genetic markers described hereinalso were used to identify the coding sequence associated with thelocus. The gene was identified as quinolinate synthase (QS); the nucleicacid sequence of the cDNA is shown in SEQ ID NO: 7, while the encodedpolypeptide is shown in SEQ ID NO: 8. Based on this discovery, the levelof expression and/or the function of QS can be modulated in N. tabacum,thereby modulating the amount of TSNAs, particularly NAT, in curedtobacco and the resulting tobacco products.

Nucleic Acids and Polypeptides

A nucleic acid encoding wild type quinolinate synthase is providedherein (see, for example, SEQ ID NO: 7). As used herein, nucleic acidscan include DNA and RNA, and includes nucleic acids that contain one ormore nucleotide analogs or backbone modifications. A nucleic acid can besingle stranded or double stranded, which usually depends upon itsintended use. The wild type quinolinate synthase nucleic acid providedherein encodes a wild type quinolinate synthase polypeptide (see, forexample, SEQ ID NO: 8).

Also provided are nucleic acids and polypeptides that differ from thewild type quinolinate synthase nucleic acid and polypeptide sequence(i.e., SEQ ID NO: 7 and 8, respectively). Nucleic acids and polypeptidesthat differ in sequence from SEQ ID NO: 7 and SEQ ID NO: 8 can have atleast 50% sequence identity (e.g., at least 55%, 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 7 and8, respectively.

In calculating percent sequence identity, two sequences are aligned andthe number of identical matches of nucleotides or amino acid residuesbetween the two sequences is determined. The number of identical matchesis divided by the length of the aligned region (i.e., the number ofaligned nucleotides or amino acid residues) and multiplied by 100 toarrive at a percent sequence identity value. It will be appreciated thatthe length of the aligned region can be a portion of one or bothsequences up to the full-length size of the shortest sequence. It alsowill be appreciated that a single sequence can align with more than oneother sequence and hence, can have different percent sequence identityvalues over each aligned region.

The alignment of two or more sequences to determine percent sequenceidentity can be performed using the computer program ClustalW anddefault parameters, which allows alignments of nucleic acid orpolypeptide sequences to be carried out across their entire length(global alignment). Chenna et al., 2003, Nucleic Acids Res.,31(13):3497-500. ClustalW calculates the best match between a query andone or more subject sequences, and aligns them so that identities,similarities and differences can be determined. Gaps of one or moreresidues can be inserted into a query sequence, a subject sequence, orboth, to maximize sequence alignments. For fast pairwise alignment ofnucleic acid sequences, the default parameters can be used (i.e., wordsize: 2; window size: 4; scoring method: percentage; number of topdiagonals: 4; and gap penalty: 5); for an alignment of multiple nucleicacid sequences, the following parameters can be used: gap openingpenalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes.For fast pairwise alignment of polypeptide sequences, the followingparameters can be used: word size: 1; window size: 5; scoring method:percentage; number of top diagonals: 5; and gap penalty: 3. For multiplealignment of polypeptide sequences, the following parameters can beused: weight matrix: blosum; gap opening penalty: 10.0; gap extensionpenalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro,Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gappenalties: on. ClustalW can be run, for example, at the Baylor Collegeof Medicine Search Launcher website or at the European BioinformaticsInstitute website on the World Wide Web.

Changes can be introduced into a nucleic acid molecule (e.g., SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, or 49), thereby leading to changes in the aminoacid sequence of the encoded polypeptide (e.g., SEQ ID NOs: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, or 50). For example, changes can be introduced into nucleic acidcoding sequences using mutagenesis (e.g., site-directed mutagenesis,PCR-mediated mutagenesis) or by chemically synthesizing a nucleic acidmolecule having such changes. Such nucleic acid changes can lead toconservative and/or non-conservative amino acid substitutions at one ormore amino acid residues. A “conservative amino acid substitution” isone in which one amino acid residue is replaced with a different aminoacid residue having a similar side chain, and a “non-conservative aminoacid substitution” is one in which an amino acid residue is replacedwith an amino acid residue that does not have a similar side chain. See,for example, Dayhoff et al. (1978, in Atlas of Protein Sequence andStructure, 5(Suppl. 3):345-352), which provides frequency tables foramino acid substitutions.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is free of sequences that naturally flank one or both endsof the nucleic acid in the genome of the organism from which theisolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNAfragment produced by PCR or restriction endonuclease digestion). Such anisolated nucleic acid molecule is generally introduced into a vector(e.g., a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule, discussed inmore detail below. In addition, an isolated nucleic acid molecule caninclude an engineered nucleic acid molecule such as a recombinant or asynthetic nucleic acid molecule.

As used herein, a “purified” polypeptide is a polypeptide that has beenseparated or purified from cellular components that naturally accompanyit. Typically, the polypeptide is considered “purified” when it is atleast 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dryweight, free from the polypeptides and naturally occurring moleculeswith which it is naturally associated. Since a polypeptide that ischemically synthesized is, by nature, separated from the components thatnaturally accompany it, a synthetic polypeptide is “purified.”

Nucleic acids can be isolated using techniques routine in the art. Forexample, nucleic acids can be isolated using any method including,without limitation, recombinant nucleic acid technology, and/or thepolymerase chain reaction (PCR). General PCR techniques are described,for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler,Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleicacid techniques include, for example, restriction enzyme digestion andligation, which can be used to isolate a nucleic acid. Isolated nucleicacids also can be chemically synthesized, either as a single nucleicacid molecule or as a series of oligonucleotides.

Polypeptides can be purified from natural sources (e.g., a biologicalsample) by known methods such as DEAE ion exchange, gel filtration, andhydroxyapatite chromatography. A polypeptide also can be purified, forexample, by expressing a nucleic acid in an expression vector. Inaddition, a purified polypeptide can be obtained by chemical synthesis.The extent of purity of a polypeptide can be measured using anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

A vector containing a nucleic acid (e.g., a nucleic acid that encodes apolypeptide) also is provided. Vectors, including expression vectors,are commercially available or can be produced by recombinant DNAtechniques routine in the art. A vector containing a nucleic acid canhave expression elements operably linked to such a nucleic acid, andfurther can include sequences such as those encoding a selectable marker(e.g., an antibiotic resistance gene). A vector containing a nucleicacid can encode a chimeric or fusion polypeptide (i.e., a polypeptideoperatively linked to a heterologous polypeptide, which can be at eitherthe N-terminus or C-terminus of the polypeptide). Representativeheterologous polypeptides are those that can be used in purification ofthe encoded polypeptide (e.g., 6×His tag, glutathione S-transferase(GST))

Expression elements include nucleic acid sequences that direct andregulate expression of nucleic acid coding sequences. One example of anexpression element is a promoter sequence. Expression elements also caninclude introns, enhancer sequences, response elements, or inducibleelements that modulate expression of a nucleic acid. Expression elementscan be of bacterial, yeast, insect, mammalian, or viral origin, andvectors can contain a combination of elements from different origins. Asused herein, operably linked means that a promoter or other expressionelement(s) are positioned in a vector relative to a nucleic acid in sucha way as to direct or regulate expression of the nucleic acid (e.g.,in-frame). Many methods for introducing nucleic acids into host cells,both in vivo and in vitro, are well known to those skilled in the artand include, without limitation, electroporation, calcium phosphateprecipitation, polyethylene glycol (PEG) transformation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transfer.

Vectors as described herein can be introduced into a host cell. As usedherein, “host cell” refers to the particular cell into which the nucleicacid is introduced and also includes the progeny or potential progeny ofsuch a cell. A host cell can be any prokaryotic or eukaryotic cell. Forexample, nucleic acids can be expressed in bacterial cells such as E.coli, or in insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

Nucleic acids can be detected using any number of amplificationtechniques (see, e.g., PCR Primer: A Laboratory Manual, 1995,Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159;and 4,965,188) with an appropriate pair of oligonucleotides (e.g.,primers). A number of modifications to the original PCR have beendeveloped and can be used to detect a nucleic acid.

Nucleic acids also can be detected using hybridization. Hybridizationbetween nucleic acids is discussed in detail in Sambrook et al. (1989,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY; Sections 7.37-7.57, 9.47-9.57,11.7-11.8, and 11.45-11.57). Sambrook et al. discloses suitable Southernblot conditions for oligonucleotide probes less than about 100nucleotides (Sections 11.45-11.46). The Tm between a sequence that isless than 100 nucleotides in length and a second sequence can becalculated using the formula provided in Section 11.46. Sambrook et al.additionally discloses Southern blot conditions for oligonucleotideprobes greater than about 100 nucleotides (see Sections 9.47-9.54). TheTm between a sequence greater than 100 nucleotides in length and asecond sequence can be calculated using the formula provided in Sections9.50-9.51 of Sambrook et al.

The conditions under which membranes containing nucleic acids areprehybridized and hybridized, as well as the conditions under whichmembranes containing nucleic acids are washed to remove excess andnon-specifically bound probe, can play a significant role in thestringency of the hybridization. Such hybridizations and washes can beperformed, where appropriate, under moderate or high stringencyconditions. For example, washing conditions can be made more stringentby decreasing the salt concentration in the wash solutions and/or byincreasing the temperature at which the washes are performed. Simply byway of example, high stringency conditions typically include a wash ofthe membranes in 0.2×SSC at 65° C.

In addition, interpreting the amount of hybridization can be affected,for example, by the specific activity of the labeled oligonucleotideprobe, by the number of probe-binding sites on the template nucleic acidto which the probe has hybridized, and by the amount of exposure of anautoradiograph or other detection medium. It will be readily appreciatedby those of ordinary skill in the art that although any number ofhybridization and washing conditions can be used to examinehybridization of a probe nucleic acid molecule to immobilized targetnucleic acids, it is more important to examine hybridization of a probeto target nucleic acids under identical hybridization, washing, andexposure conditions. Preferably, the target nucleic acids are on thesame membrane.

A nucleic acid molecule is deemed to hybridize to a nucleic acid but notto another nucleic acid if hybridization to a nucleic acid is at least5-fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,50-fold, or 100-fold) greater than hybridization to another nucleicacid. The amount of hybridization can be quantitated directly on amembrane or from an autoradiograph using, for example, a PhosphorImageror a Densitometer (Molecular Dynamics, Sunnyvale, Calif.).

Polypeptides can be detected using antibodies. Techniques for detectingpolypeptides using antibodies include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Anantibody can be polyclonal or monoclonal. An antibody having specificbinding affinity for a polypeptide can be generated using methods wellknown in the art. The antibody can be attached to a solid support suchas a microtiter plate using methods known in the art. In the presence ofa polypeptide, an antibody-polypeptide complex is formed.

Detection (e.g., of an amplification product, a hybridization complex,or a polypeptide) is usually accomplished using detectable labels. Theterm “label” is intended to encompass the use of direct labels as wellas indirect labels. Detectable labels include enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials.

Plants Having Reduced Amounts of NAT in Leaf and Methods of Making

Tobacco hybrids, varieties, lines, or cultivars are provided that have amutation in an endogenous quinolinate synthase nucleic acid (e.g., SEQID NO: 7). The quinolinate synthase A protein (NadA) is a component ofthe quinolinate synthase complex, which includes quinolinate synthase Aprotein and L-aspartate oxidase protein. L-aspartate oxidase (NadB) isan FAD-dependent enzyme catalyzing the oxidation of L-aspartate toiminoaspartate. Iminoaspartate is then condensed with DHAP to formquinolinate under the action of quinolinate synthase A, the gene productof nadA.

As described herein, leaf from plants having a mutation in one or moreof the endogenous nucleic acids (e.g., SEQ ID NO: 7) can exhibit areduced amount of alkaloids (e.g., anatabine) and/or TSNAs (e.g., NAT)compared to leaf from a plant that lacks the mutation. In addition, leaffrom plants having a mutation in an endogenous quinolinate synthasenucleic acid (e.g., SEQ ID NO: 7) can exhibit a reduced amount ofalkaloids or TSNAs (e.g., compared to leaf from a plant lacking themutation).

Methods of detecting one or more alkaloids and one or more TSNAs, andmethods of determining the amount of one or more alkaloids and one ormore TSNAs, are known in the art. For example, high performance liquidchromatography (HPLC)-mass spectroscopy (MS) (HPLC-MS) or highperformance thin layer chromatography (HPTLC) can be used to detect thepresence of and/or determine the amount of alkaloids or TSNAs. Inaddition, any number of chromatography methods (e.g., gaschromatography/thermal energy analysis (GC/TEA), liquidchromatography/mass spectrometry (LC/MS), and ion chromatography (IC))can be used to detect the presence of and/or determine the amount ofalkaloids or TSNAs.

Methods of making a tobacco plant having a mutation are known in theart. Mutations can be random mutations or targeted mutations. For randommutagenesis, cells (e.g., Nicotiana tabacum cells) typically aremutagenized using, for example, a chemical mutagen or ionizingradiation. Representative chemical mutagens include, without limitation,nitrous acid, sodium azide, acridine orange, ethidium bromide, and ethylmethane sulfonate (EMS), while representative ionizing radiationincludes, without limitation, x-rays, gamma rays, fast neutronirradiation, and UV irradiation. The dosage of the mutagenic chemical orradiation is determined experimentally for each type of plant tissuesuch that a mutation frequency is obtained that is below a thresholdlevel characterized by lethality or reproductive sterility. The numberof M₁ generation seed or the size of M₁ plant populations resulting fromthe mutagenic treatments are estimated based on the expected frequencyof mutations. For targeted mutagenesis, representative technologiesinclude TALEN (see, for example, Li et al., 2011, Nucleic Acids Res.,39(14):6315-25) or zinc-finger (see, for example, Wright et al., 2005,The Plant J., 44:693-705). Whether random or targeted, a mutation can bea point mutation, an insertion, a deletion, a substitution, orcombinations thereof.

As discussed herein, one or more nucleotides can be mutated to alter theexpression and/or function of the encoded polypeptide, relative to theexpression and/or function of the corresponding wild type polypeptide.It will be appreciated, for example, that a mutation in one or more ofthe highly conserved regions would likely alter polypeptide function,while a mutation outside of those conserved regions would likely havelittle to no effect on polypeptide function. In addition, a mutation ina single nucleotide can create a stop codon, which would result in atruncated polypeptide and, depending on the extent of truncation,loss-of-function.

Preferably, a mutation in one of the novel nucleic acids disclosedherein results in reduced or even complete elimination of quinolinatesynthase activity in a tobacco plant comprising the mutation. Suitabletypes of mutations in a quinolinate synthase coding sequence include,without limitation, insertions of nucleotides, deletions of nucleotides,or transitions or transversions in the wild-type quinolinate synthasecoding sequence. Mutations in the coding sequence can result ininsertions of one or more amino acids, deletions of one or more aminoacids, and/or non-conservative amino acid substitutions in the encodedpolypeptide. In some cases, the coding sequence comprises more than onemutation or more than one type of mutation.

Insertion or deletion of amino acids in a coding sequence, for example,can disrupt the conformation of the encoded polypeptide. Amino acidinsertions or deletions also can disrupt sites important for recognitionof the binding ligand (e.g., DHAP). It is known in the art that theinsertion or deletion of a larger number of contiguous amino acids ismore likely to render the gene product non-functional, compared to asmaller number of inserted or deleted amino acids. In addition, one ormore mutations (e.g., a point mutation) can change the localization ofthe transporter polypeptide, introduce a stop codon to produce atruncated polypeptide, or disrupt an active site or domain (e.g., acatalytic site or domain, a binding site or domain) within thepolypeptide.

Non-conservative amino acid substitutions can replace an amino acid ofone class with an amino acid of a different class. Non-conservativesubstitutions can make a substantial change in the charge orhydrophobicity of the gene product. Non-conservative amino acidsubstitutions can also make a substantial change in the bulk of theresidue side chain, e.g., substituting an alanine residue for anisoleucine residue. Examples of non-conservative substitutions include abasic amino acid for a non-polar amino acid, or a polar amino acid foran acidic amino acid.

Following mutagenesis, M₀ plants are regenerated from the mutagenizedcells and those plants, or a subsequent generation of that population(e.g., M₁, M₂, M₃, etc.), can be screened for a mutation in aquinolinate synthase nucleic acid sequence (e.g., SEQ ID NO: 7).Screening for plants carrying a mutation in a sequence of interest canbe performed using methods routine in the art (e.g., hybridization,amplification, combinations thereof) or by evaluating the phenotype(e.g., detecting and/or determining the amount of anatabine and/or NATin the roots and/or the leaf). Generally, the presence of a mutation ina quinolinate synthase nucleic acid (e.g., SEQ ID NO: 7) results in areduction of anatabine in the leaf of the mutant plants and NAT in thecured leaf of the mutant plants compared to a corresponding plant (e.g.,having the same varietal background) lacking the mutation.

As used herein, “reduced” or “reduction” refers to a decrease (e.g., astatistically significant decrease) in the amount of anatabine intobacco leaf, either green or cured, and/or NAT in cured leaf by atleast about 5% up to about 95% (e.g., about 5% to about 10%, about 5% toabout 20%, about 5% to about 50%, about 5% to about 75%, about 10% toabout 25%, about 10% to about 50%, about 10% to about 90%, about 20% toabout 40%, about 20% to about 60%, about 20% to about 80%, about 25% toabout 75%, about 50% to about 75%, about 50% to about 85%, about 50% toabout 95%, and about 75% to about 95%) relative to similarly-treatedleaf (e.g., green or cured) from a tobacco plant lacking the mutation.As used herein, statistical significance refers to a p-value of lessthan 0.05, e.g., a p-value of less than 0.025 or a p-value of less than0.01, using an appropriate measure of statistical significance, e.g., aone-tailed two sample t-test.

An M₁ tobacco plant may be heterozygous for a mutant allele and exhibita wild type phenotype. In such cases, at least a portion of the firstgeneration of self-pollinated progeny of such a plant exhibits a wildtype phenotype. Alternatively, an M₁ tobacco plant may have a mutantallele and exhibit a mutant phenotype. Such plants may be heterozygousand exhibit a mutant phenotype due to a phenomenon such as dominantnegative suppression, despite the presence of the wild type allele, orsuch plants may be homozygous due to independently induced mutations inboth alleles.

A tobacco plant carrying a mutant allele can be used in a plant breedingprogram to create novel and useful cultivars, lines, varieties andhybrids. Thus, in some embodiments, an M₁, M₂, M₃ or later generationtobacco plant containing at least one mutation is crossed with a secondNicotiana tabacum plant, and progeny of the cross are identified inwhich the mutation(s) is present. It will be appreciated that the secondNicotiana tabacum plant can be one of the species and varietiesdescribed herein. It will also be appreciated that the second Nicotianatabacum plant can contain the same mutation as the plant to which it iscrossed, a different mutation, or be wild type at the locus.Additionally or alternatively, a second tobacco line can exhibit aphenotypic trait such as, for example, disease resistance, high yield,high grade index, curability, curing quality, mechanical harvesting,holding ability, leaf quality, height, plant maturation (e.g., earlymaturing, early to medium maturing, medium maturing, medium to latematuring, or late maturing), stalk size (e.g., small, medium, or large),and/or leaf number per plant (e.g., a small (e.g., 5-10 leaves), medium(e.g., 11-15 leaves), or large (e.g., 16-21) number of leaves).

Breeding is carried out using known procedures. DNA fingerprinting, SNPor similar technologies may be used in a marker-assisted selection (MAS)breeding program to transfer or breed mutant alleles into othertobaccos, as described herein. Progeny of the cross can be screened fora mutation using methods described herein, and plants having a mutationin a quinolinate synthase nucleic acid sequence (e.g., SEQ ID NO: 7) canbe selected. For example, plants in the F₂ or backcross generations canbe screened using a marker developed from a sequence described herein ora fragment thereof, using one of the techniques listed herein. Leaf(green or cured, as appropriate) from progeny plants also can bescreened for the amount of anatabine and/or NAT, and those plants havingreduced amounts, compared to a corresponding plant that lacks themutation, can be selected. Plants identified as possessing the mutantallele and/or the mutant phenotype can be backcrossed or self-pollinatedto create a second population to be screened. Backcrossing or otherbreeding procedures can be repeated until the desired phenotype of therecurrent parent is recovered.

Successful crosses yield F₁ plants that are fertile and that can bebackcrossed with one of the parents if desired. In some embodiments, aplant population in the F₂ generation is screened for the mutation orvariant gene expression using standard methods (e.g., PCR with primersbased upon the nucleic acid sequences disclosed herein). Selected plantsare then crossed with one of the parents and the first backcross (BC₁)generation plants are self-pollinated to produce a BC₁F₂ population thatis again screened for variant gene expression. The process ofbackcrossing, self-pollination, and screening is repeated, for example,at least four times until the final screening produces a plant that isfertile and reasonably similar to the recurrent parent. This plant, ifdesired, is self-pollinated and the progeny are subsequently screenedagain to confirm that the plant contains the mutation and exhibitsvariant gene expression. Breeder's seed of the selected plant can beproduced using standard methods including, for example, field testing,confirmation of the null condition, and/or chemical analyses of leaf(e.g., cured leaf) to determine the level of anatabine and/or NAT.

The result of a plant breeding program using the mutant tobacco plantsdescribed herein are novel and useful cultivars, varieties, lines, andhybrids. As used herein, the term “variety” refers to a population ofplants that share constant characteristics which separate them fromother plants of the same species. A variety is often, although notalways, sold commercially. While possessing one or more distinctivetraits, a variety is further characterized by a very small overallvariation between individual with that variety. A “pure line” varietymay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques. A “line,” as distinguished from a variety, mostoften denotes a group of plants used non-commercially, for example, inplant research. A line typically displays little overall variationbetween individuals for one or more traits of interest, although theremay be some variation between individuals for other traits.

A variety can be essentially derived from another line or variety. Asdefined by the International Convention for the Protection of NewVarieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov. 10,1972, On Oct. 23, 1978, and on Mar. 19, 1991), a variety is “essentiallyderived” from an initial variety if: a) it is predominantly derived fromthe initial variety, or from a variety that is predominantly derivedfrom the initial variety, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of the initial variety; b) it is clearly distinguishablefrom the initial variety; and c) except for the differences which resultfrom the act of derivation, it confirms to the initial variety in theexpression of the essential characteristics that result from thegenotype or combination of genotypes of the initial variety. Essentiallyderived varieties can be obtained, for example, by the selection of anatural or induced mutant, a somaclonal variant, a variant individualplant from the initial variety, backcrossing, or transformation.

Hybrid tobacco varieties can be produced by preventing self-pollinationof female parent plants (i.e., seed parents) of a first variety,permitting pollen from male parent plants of a second variety tofertilize the female parent plants, and allowing F₁ hybrid seeds to formon the female plants. Self-pollination of female plants can be preventedby emasculating the flowers at an early stage of flower development.Alternatively, pollen formation can be prevented on the female parentplants using a form of male sterility. For example, male sterility canbe produced by cytoplasmic male sterility (CMS), nuclear male sterility,genetic male sterility, molecular male sterility wherein a transgeneinhibits microsporogenesis and/or pollen formation, orself-incompatibility. Female parent plants containing CMS areparticularly useful. In embodiments in which the female parent plantsare CMS, the male parent plants typically contain a fertility restorergene to ensure that the F₁ hybrids are fertile. In other embodiments inwhich the female parents are CMS, male parents can be used that do notcontain a fertility restorer. F₁ hybrids produced from such parents aremale sterile. Male sterile hybrid seed can be interplanted with malefertile seed to provide pollen for seed-set on the resulting malesterile plants.

Varieties and lines described herein can be used to form single-crosstobacco F₁ hybrids. In such embodiments, the plants of the parentvarieties can be grown as substantially homogeneous adjoiningpopulations to facilitate natural cross-pollination from the male parentplants to the female parent plants. The F₂ seed formed on the femaleparent plants is selectively harvested by conventional means. One alsocan grow the two parent plant varieties in bulk and harvest a blend ofF₁ hybrid seed formed on the female parent and seed formed upon the maleparent as the result of self-pollination. Alternatively, three-waycrosses can be carried out wherein a single-cross F₁ hybrid is used as afemale parent and is crossed with a different male parent. As anotheralternative, double-cross hybrids can be created wherein the F₁ progenyof two different single-crosses are themselves crossed.Self-incompatibility can be used to particular advantage to preventself-pollination of female parents when forming a double-cross hybrid.

As used herein, the term “cultivar” refers to a uniform variety, strainor race of plant selected for desirable characteristics that aremaintained by vegetative propagation or by inbred seed.

The tobacco plants used in the methods described herein can be a Burleytype, a dark type, a flue-cured type, a Maryland type, or an Orientaltype. The tobacco plants used in the methods described herein typicallyare from N. tabacum, and can be from any number of N. tabacum varieties.A variety can be BU 64, CC 101, CC 200, CC 13, CC 27, CC 33, CC 35, CC37, CC 65, CC 67, CC 301, CC 400, CC 500, CC 600, CC 700, CC 800, CC900, CC 1063, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CU 263,DF911, Galpao tobacco, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737,GL 939, GL 973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K358, K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326,NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA 116,VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17, KY 171,KY 907, KY907LC, KTY14×L8 LC, Little Crittenden, McNair 373, McNair 944,msKY 14×L8, Narrow Leaf Madole, NC 100, NC 102, NC 2000, NC 291, NC 297,NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH129, NC 2002, Neal Smith Madole, OXFORD 207, ‘Perique’ tobacco, PVH03,PVH09, PVH19, PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81,RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179,Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, SpeightG-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, TI 1406, TI1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN D94, TN D950, TR(Tom Rosson) Madole, VA 309, or VA359.

In addition to mutation, another way in which the amount of anatabineand NAT in tobacco leaf can be reduced is to use inhibitory RNAs (e.g.,RNAi). Therefore, transgenic tobacco plants are provided that contain atransgene encoding at least one RNAi molecule, which, when transcribed,silences an endogenous quinolinate synthase nucleic acid (e.g., SEQ IDNO: 7). It would be understood in the art that “silencing” can refer tocomplete elimination or essentially complete elimination of thequinolinate synthase mRNA, resulting in 100% or essentially 100%reduction (e.g., greater than 95% reduction; e.g., greater than 96%,97%, 98% or 99% reduction) in the amount of quinolinate synthasepolypeptide; silencing also can refer to partial elimination of thequinolinate synthase mRNA (e.g., eliminating about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50% or more of the quinolinate synthase mRNA),resulting in a reduction (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50% or more, but not complete elimination) in the amount ofthe quinolinate synthase polypeptide. As described herein, leaf fromsuch transgenic plants exhibit a reduced amount of anatabine and NAT(e.g., compared to leaf from a plant lacking or not transcribing theRNAi).

RNAi technology is known in the art and is a very effective form ofpost-transcriptional gene silencing. RNAi molecules typically contain anucleotide sequence (e.g., from about 18 to about 50 nucleotides inlength) that is complementary to the target gene in both the sense andantisense orientations. The sense and antisense strands can be connectedby a short “loop” sequence (e.g., about 5 to about 20 nucleotides inlength) and transcribed in a single transcript, or the sense andantisense strands can be delivered to and transcribed in the targetcells on separate vectors or constructs. A number of companies offerRNAi design and synthesis services (e.g., Life Technologies, AppliedBiosystems).

The RNAi molecule can be transcribed using a plant expression vector.The RNAi molecule typically is at least 25 nucleotides in length and hasat least 91% sequence identity (e.g., at least 95%, 96%, 97%, 98% or 99%sequence identity) to a quinolinate synthase nucleic acid sequencedisclosed herein (e.g., SEQ ID NO: 7) or hybridizes under stringentconditions to a quinolinate synthase nucleic acid sequence disclosedherein (e.g., SEQ ID NO: 7). Hybridization under stringent conditions isdescribed above.

Methods of introducing a nucleic acid (e.g., a heterologous nucleicacid) into plant cells are known in the art and include, for example,particle bombardment, Agrobacterium-mediated transformation,microinjection, polyethylene glycol-mediated transformation (e.g., ofprotoplasts, see, for example, Yoo et al. (2007, Nature Protocols,2(7):1565-72)), liposome-mediated DNA uptake, or electroporation.Following transformation, the transgenic plant cells can be regeneratedinto transgenic tobacco plants. As described herein, transcription ofthe transgene results in leaf that exhibits a reduced amount ofanatabine or NAT in the resulting cured leaf relative to leaf from aplant not transcribing the transgene. The leaves of the regeneratedtransgenic plants can be screened for the amount of anatabine or NAT inthe resulting cured leaf, and plants having reduced amounts of anatabineor NAT in the resulting cured leaf, compared to the amount in acorresponding non-transgenic plant, can be selected for use in, forexample, a breeding program as discussed herein.

Nucleic acids that confer traits such as herbicide resistance (sometimesreferred to as herbicide tolerance), insect resistance, or stresstolerance, can also be present in the novel tobacco plants describedherein. Genes conferring resistance to a herbicide that inhibits thegrowing point or meristem, such as an imidazolinone or a sulfonylurea,can be suitable. Exemplary genes in this category encode mutant ALS andAHAS enzymes as described, for example, in U.S. Pat. Nos. 5,767,366 and5,928,937. U.S. Pat. Nos. 4,761,373 and 5,013,659 are directed to plantsresistant to various imidazolinone or sulfonamide herbicides. U.S. Pat.No. 4,975,374 relates to plant cells and plants containing a geneencoding a mutant glutamine synthetase (GS), which is resistant toinhibition by herbicides that are known to inhibit GS, e.g.phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,162,602discloses plants resistant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides.

Genes for resistance to glyphosate also are suitable. See, for example,U.S. Pat. Nos. 4,940,835 and 4,769,061. Such genes can confer resistanceto glyphosate herbicidal compositions, including, without limitation,glyphosate salts such as the trimethylsulphonium salt, theisopropylamine salt, the sodium salt, the potassium salt and theammonium salt. See, e.g., U.S. Pat. Nos. 6,451,735 and 6,451,732. Genesfor resistance to phosphono compounds such as glufosinate ammonium orphosphinothricin, and pyridinoxy or phenoxy propionic acids andcyclohexones also are suitable. See, e.g., U.S. Pat. Nos. 5,879,903;5,276,268; and 5,561,236; and European Application No. 0 242 246.

Other suitable herbicides include those that inhibit photosynthesis,such as a triazine and a benzonitrile (nitrilase). See U.S. Pat. No.4,810,648. Other suitable herbicides include 2,2-dichloropropionic acid,sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylureaherbicides, triazolopyrimidine herbicides, s-triazine herbicides andbromoxynil. Also suitable are herbicides that confer resistance to aprotox enzyme. See, e.g., U.S. Pat. No. 6,084,155 and US 20010016956.

A number of genes are available that confer resistance to insects, forexample, insects in the order Lepidoptera. Exemplary genes include thosethat encode truncated CrylA(b) and CrylA(c) toxins. See, e.g., genesdescribed in U.S. Pat. Nos. 5,545,565; 6,166,302; and 5,164,180. Seealso, Vaeck et al., 1997, Nature, 328:33-37 and Fischhoff et al., 1987,Nature Biotechnology, 5:807-813. Particularly useful are genes encodingtoxins that exhibit insecticidal activity against Manduca sexta (tobaccohornworm); Heliothis virescens Fabricius (tobacco budworm) and/or S.litura Fabricius (tobacco cutworm).

Plants Having Increased Amounts of Anatabine in Leaf and Methods ofMaking

The sequences described herein can be overexpressed in plants in orderto increase the amount of anatabine in the leaf. Therefore, transgenictobacco plants, or leaf from such plants, are provided that include aplant expression vector. A plant expression vector typically includes aquinolinate synthase nucleic acid molecule described herein (e.g., SEQID NO: 7) or a functional fragment thereof under control of a promoterthat is able to drive expression in plants (e.g., a plant promoter). Asdiscussed herein, a nucleic acid molecule used in a plant expressionvector can have a different sequence than a sequence described herein,which can be expressed as a percent sequence identity (e.g., relative toSEQ ID NO: 7) or based on the conditions under which the sequencehybridizes to SEQ ID NO: 7.

As an alternative to using a full-length sequence, a portion of thesequence can be used that encodes a polypeptide fragment having thedesired functionality (referred to herein as a “functional fragment”).When used with respect to nucleic acids, it would be appreciated that itis not the nucleic acid fragment that possesses functionality but theencoded polypeptide fragment. Based on the disclosure herein, one ofskill in the art can predict the portion(s) of a polypeptide (e.g., oneor more domains) that may impart the desired functionality.

Following transformation, the transgenic tobacco cells can beregenerated into transgenic tobacco plants. The leaves of theregenerated tobacco plants can be screened for the amount of anatabine,and plants having increased amounts of anatabine, compared to the amountin a corresponding non-transgenic plant, can be selected and used, forexample, in a breeding program as discussed herein. Expression of thenucleic acid molecule or a functional fragment thereof may result inleaf that exhibits an increased amount of anatabine compared to leaffrom a tobacco plant that does not express the nucleic acid molecule orfunctional fragment thereof.

Genetic Markers of Low-/Ultra-Low-Anatabine Trait in Tobacco

Although coding sequences (i.e., the sequences that encode a protein)generally are well-conserved within a species, non-coding regions tendto accumulate polymorphism, and, therefore, can vary between individualsof the same species. Such regions provide the basis for genetic markers.In general, any differentially-inherited nucleic acid polymorphism thatsegregates among progeny is a potential genetic marker. The genomicvariability that results in a genetic marker can be of any origin,including insertions, deletions, duplications, repetitive elements,point mutations, recombination events, and the presence and sequence oftransposable elements.

In general, the identification of markers for MAS application involvesdetermining the phenotype of the trait(s) of interest and genotyping thesegregating population of progenies using polymorphic markers andgenetic mapping of the desired trait. Details of mapping are describedelsewhere herein. Polymorphic loci in the vicinity of the mapped traitare chosen as potential markers (typically, a marker locus closest tothe locus of interest is a preferred marker). Linkage analysis is thenused to determine which polymorphic marker allele sequence demonstratesa statistical likelihood of co-segregation with the desirable phenotype(thus, a “marker allele”). It is then possible to use this marker forrapid, accurate screening of plant lines for the marker allele withoutthe need to grow the plants through their life cycle and awaitphenotypic evaluations.

Numerous methods for detecting genetic markers also arewell-established. Markers corresponding to genetic polymorphisms betweenmembers of a population can be detected by numerous methods that arewell-established in the art such as, without limitation, restrictionfragment length polymorphisms, isozyme markers, allele specifichybridization (ASH), amplified variable sequences of the plant genome,self-sustained sequence replication, simple sequence repeats (SSRs),single nucleotide polymorphisms (SNPs), or amplified fragment lengthpolymorphisms (AFLPs). The genetic markers described herein are SSRs.See, for example, any of SEQ ID NOs:1-6.

Genetic markers can facilitate mapping and selection of agriculturallyimportant traits. A genetic marker that demonstrates linkagedisequilibrium with a desired trait (e.g., low- or ultra-low-anatabine)can be a useful tool for marker-assisted selection (MAS), providing ameans for rapid identification of desirable individuals or lines.Introgression of one or more particular genes into a cultivar or varietyalso can be facilitated by MAS. Simply by way of example, MAS caninclude the steps of (i) creating a map of genetic markers; (ii)determining statistical associations between one or more genetic markersand a phenotype (or phenotypic variability); (iii) identifying/defininga set of genetic markers associated with the phenotype (or phenotypicvariability); and (iv) applying this information to a breeding program.

MAS can be used in plant breeding to assist in the efficient recoveryof, for example, the recurrent parent genotype following backcrossing.In marker-assisted backcrossing, progeny can be selected for the donortrait (e.g., low- or ultra-low anatabine) and then repeatedlybackcrossed to, for example, an elite line to reconstitute as much aspossible of the elite line's background.

Tobacco Products and Methods of Making

The methods described herein allow for leaf constituents in a tobaccoplant to be altered. As described herein, altering leaf constituentsrefers to reducing the amount of one or more alkaloids (e.g., anatabine)or one or more TSNAs (e.g., NAT) in the leaf or increasing the amount ofone or more alkaloids (e.g., anatabine) in the leaf. As describedherein, such methods can include mutagenesis (e.g., random or targeted)or the production of transgenic plants (using, e.g., RNAi oroverexpression).

Leaf from such tobacco (e.g., having reduced amounts of one or morealkaloids or one or more TSNAs or increased amounts of one or morealkaloids) can be cured, aged, conditioned, and/or fermented. Methods ofcuring tobacco are well known and include, for example, air curing, firecuring, flue curing and sun curing. Aging also is known and typically iscarried out in a wooden drum (e.g., a hogshead) or cardboard cartons incompressed conditions for several years (e.g., 2 to 5 years), at amoisture content of from about 10% to about 25% (see, for example, U.S.Pat. Nos. 4,516,590 and 5,372,149). Conditioning includes, for example,a heating, sweating or pasteurization step as described in US2004/0118422 or US 2005/0178398, while fermenting typically ischaracterized by high initial moisture content, heat generation, and a10 to 20% loss of dry weight. See, e.g., U.S. Pat. Nos. 4,528,993,4,660,577, 4,848,373 and 5,372,149. The tobacco also can be furtherprocessed (e.g., cut, expanded, blended, milled or comminuted), ifdesired, and used in a tobacco product.

Tobacco products are known in the art and include any product made orderived from tobacco that is intended for human consumption, includingany component, part, or accessory of a tobacco product. Representativetobacco products include, without limitation, smokeless tobaccoproducts, tobacco-derived nicotine products (e.g., tobacco-derivednicotine pieces for use in the mouth), cigarillos, non-ventilated recessfilter cigarettes, vented recess filter cigarettes, cigars, snuff, pipetobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaftobacco, shredded tobacco, cut tobacco, electronic cigarettes,electronic cigars, electronic cigarillos, and e-vapor devices.Representative smokeless tobacco products include, for example, chewingtobacco, snus, pouches, films, tablets, coated dowels, rods, and thelike. Representative cigarettes and other smoking articles include, forexample, smoking articles that include filter elements or rod elements,where the rod element of a smokeable material can include cured tobaccowithin a tobacco blend. In addition to the reduced-alkaloid tobacco, thereduced-TSNA tobacco, or the increased-alkaloid tobacco describedherein, tobacco products also can include other ingredients such as,without limitation, binders, plasticizers, stabilizers, and/orflavorings. See, for example, US 2005/0244521, US 2006/0191548, US2012/0024301, US 2012/0031414, and US 2012/0031416 for examples oftobacco products.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1—Mutagenesis

Since the biosynthetic pathway of anatabine and its associated genes isnot completely known, a novel genetic variation was created in apopulation of tobacco plants to identify plants that have asignificantly reduced ability to biosynthesize anatabine. These plantsvery likely have a mutated non-functional gene, critical for anatabinebiosynthesis.

A population of the Flue-cured variety “401” was used in theseexperiments. Approximately 5000 seeds were treated with 0.6% ethylmethane sulfonate and germinated. M1 plants were grown in the field andM2 seeds were collected. Fifteen hundred M2 seeds were germinated andgrown in 4-inch pots. At 50% flowering stage, plants were topped. Leafsamples were collected 2 weeks after topping and the samples screenedfor anatabine levels using high performance thin layer chromatography(HP-TLC) and gas chromatography.

After screening for alkaloids, two Flue Cured (FC) 401 ultra-lowanatabine (ULA) lines were selected for trait development. It is notedthat the amount of nicotine in both ULA lines is unchanged.

Example 2—Identification of Genetic Markers

To identify genetic marker(s) associated with the ULA trait, testcrosses of FC401 mutant #1 (MS4144) were made with variety Red Russianand the F1s were selfed to generate F2 seed. Three hundred and thirtyseven F2 plants were grown in the field and the alkaloids were analyzedindividually. Depending on the anatabine levels, the mapping populationswere grouped into ULA plants and normal plants (FIG. 1). Genomic DNAfrom each of the plants was extracted individually. To run simplesequence repeat (SSR) markers, DNA samples from 23 F2 ULA plants and 24normal anatabine plants were pooled separately.

PCR reactions were performed in 25 μl final volumes which contained25-50 ng of template DNA, 12.5 μl 2× Amplitag PCR master mix ((AppliedBiosystems [ABI]), 0.2 μM labeled primers (ABI), 1 μl 100% DMSO (FisherScientific), and 8 μl H₂O (DNase/RNase free). Thermocycling conditionsconsisted of a 15 min incubation at 95° C.; followed by 34 cycles of 1min at 94° C., 2 min at 60° C., 1 min at 72° C.; with a final reactionstep of 60° C. for 30 min. All completed PCR reactions were diluted 1:50with deionized water. Two microliters of diluted product was thencombined with 9.75 μl HiDi Formamide (ABI) and 0.25 μl GeneScan 500 LIZ(ABI) size standard. Fragment analyses were performed. Samples wereseparated using a 36 cm capillary array in an ABI 3730 DNA Analyzer.Generated amplicons were analyzed using the “Local Southern Method” andthe default analysis settings within GeneMapper v. 3.5 software (ABI).Final allele calls were standardized to an internal DNA control andbased on the ABI 3730 DNA Analyzer.

Example 3—Linkage Mapping

A representative set of 246 SSR markers were selected and assessed inmapping parental lines (FC401×Red Russian). Of the 246 markers, 239showed polymorphism among parents and were mapped and assessed for ULAtrait screening. Six of those markers showed polymorphisms among ULA andnon ULA traits and were tightly linked to the ULA trait. The six geneticmarkers are shown in Table 2, and all were found to be located onchromosome 6. Sequence information of the primer sets that were used arelisted in Table 3.

TABLE 2 SSR markers tightly linked with ULA trait SSR DesignationSSR Sequence SEQ ID NO PT61163 AGTGGCGGAGGTAGGAATTTCACCAAGAGAATTCAAA 1AAAATAAAAGTATACATGCAAAGAAGCAAACGTGATTCATCACCTAATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATACATATACATAAAAATAAAATTTGACTTTATATACATAGT GTAATTTCCCGGCTACCTCACTCTTA PT60043CCCTTTGCCACTTTACAAGAATTTTCCCTCATGTCGAG 2GGGATTCAACAGCTTTTATATATATATATATATATATAAAGGAAAAAAGTAGTTGTTATCCTATTTGCACAA PT60878AGCTCCATTTGTACTTCCGCGAGCGAGGTTCCAGACAG 3CCTATTTAATAATATATATATATATATATATATATATTCACAAATATTGTGTATATACTACATACACACACATATATAAAGTGTGCATAGTGTAAACAATAGCTCACTTATTCACATAAGACTTAAGTTTGTATAATCATCTTGGAACGTCT GTTAGAA PT61060CACAAGACCCTTCTTGGTGCTGTAGAAATTGAAAGAAAT 4GCAAAGGCTTTTTTTCTACTCTATCCTGTATTTCAATGTTTGGTGTGATTAAGCTATTATGCAGAATTTTGTATAGCAAGGAACCATATATATATATATATATATATATATATATATATATACTAGACAGGGTTGCTGCCCAAAATATTAGGCGTGAAGCCTGTCTTGGTGGCAATATCAATAGTGCTCCGCCCA AAATATTAGGCGTGAAGCCTGTCTTPT60925 CAGAGATTACACCCATTGTGCTTCTTTTTAAAGGAGTAAA 5GTGGGAAAAAATCACCAAATACTTGAATAAAGACAGAGAATAACCCCTCTCTCTCTCTCTCTCTCACACACACTCCTTTTTCGGCATTTATATTATAGAGCACACAGACAATAGATCTATGGGTGAGAAAGATACTATTACTACCTACATTGACGGCGG PT50801TGTCTCAACTTCTTGTCAATTGCTAATCACTCTTTTTATG 6GAATTGACATGCTACATATATATATATATATATATATATATTACTTTTCTTAGCAAATATATTTATAAGTATCGCAGGAT GACATAATAAGG

TABLE 3 Primers used to detect the SSR markers SEQ SEQ ID ID PrimerForward Primer NO Reverse Primer NO P161163 AGTGGCGGAGGTAGGAATTT 9TAAGAGTGAGGTAGCCGGGA 10 P160043 CCCTTTGCCACTTTACAAGAA 11TTGTGCAAATAGGATAACAACTACTTT 12 P160878 AGCTCCATTTGTACTTCCGC 13TTCTAACAGACGTTCCAAGATGA 14 P161060 CACAAGACCCTTCTTGGTGC 15ACGTGGAGTGGAGAAACCAG 16 P160925 CAGAGATTACACCCATTGTGCT 17CCGCCGTCAATGTAGGTAGT 18 P150801 TGTCTCAACTTCTTGTCAATTGCT 19CCTTATTATGTCATCCTGCGA 20

To evaluate the nature of the mutant gene in the FC401 ULA mutant #2, across between the FC401 mutant #1 and FC401 mutant #2 was made and thealkaloids were analyzed in the F1 generation. Even though they are twoindependent lines, the anatabine profile of F1 plants showed that theyare mutants for the same gene (FIGS. 2A and 2B). Therefore, anatabineprofile and genetic marker analysis indicate that a single recessivegene is critical for anatabine biosynthesis and is located on geneticlinkage map 6.

Example 4—Identifying Candidate Genes

A list of candidate target genes that may be responsible for the lowanatabine trait were generated from the following three different setsof gene expression data, as follows.

A) Methyl jasmonate up-regulated genes: Six samples of Nicotiana tabacumcultivar Bright Yellow—2, in two treatment groups of 3 samples each, areexamined for gene expression. The first group consists of mock-treatedcontrol samples (CTR) obtained at 0, 48 and 72 hrs post-treatment. Thesecond group consists of samples treated with methyl jasmonate andobtained at the same time points. Samples are analyzed by microarray(Cogenics), and log-ratio p-values and fold-changes for each probe aredetermined by Rosetta Resolver using the Agilent/Intensity-PairwiseRatio Builder. These values are used to compare with the datasets fromB) and C).

B) Topping up-regulated root expression genes: Forty eighty tobaccoplants of the VA359 tobacco variety are grown in 10-inch pots. At 50%flowering, plants are topped; root samples are collected just beforetopping and at 30 mins, 2 hrs, 6 hrs, 12 hrs, 24 hrs, 72 hrs and 1 weekpost-topping. Six plants per time point are used for sample collection.Total RNA is isolated individually, equal amounts are pooled for eachtime point, and the samples hybridized with the tobacco microarray(Nimblegen). Log-ratio p-values and fold-changes for each array aredetermined by Rosetta Resolver using the Agilent/Intensity-PairwiseRatio Builder. These values are used to compare with datasets from A)and C).

C) Methyl jasmonate up-regulated proteins: Triplicate samples of BY2cells are mock-treated or treated with methyl jasmonate for 12, 24, and48 hrs. Proteins from these samples are extracted and proteomic analysisis done by Biognosys Inc. Briefly, total proteins from the samples areused to produce a master list of all possible proteins that can bedetected by the assay. Individual samples are then subjected toquantitative mass spectroscopy, and differential protein expressionlevels among each sample are generated. These values are used to comparegene expression profiles among the datasets from A) and B).

Example 5—Screening and Cloning Candidate Genes

Three tobacco lines, FC401 wild type (Wt); FC40-M207 mutant line fourthgeneration (M4) and FC401-M544 mutant line fourth generation (M4) wereused for candidate gene screening. Low anatabine traits were confirmedfor the two tobacco mutant lines (M207 and M544) in root and leaf beforescreening (see FIG. 3).

RNA was extracted from root tissues of wild type (Wt) FC401, M207 andM544 with RNeasy Plus Mini kit from Quiagen Inc. following themanufacturer's protocol. cDNA libraries were prepared from the RNAsusing In-Fusion® SMARTer® Directional cDNA Library Construction Kit fromClontech Inc. cDNA libraries were diluted to 100 ng/μl and used as thetemplate for candidate gene PCR screening.

PCR amplifications were performed in 50 μl final volumes that contained50-100 ng of template DNA (i.e., the cDNA library) and 0.2 μM of primers(Fisher Scientific) using the Platinum® Taq DNA Polymerase High Fidelitykit (Life Technology Inc.). Thermocycling conditions included a 5 minincubation at 94° C.; followed by 34 cycles of 30 seconds at 94° C., 30seconds at 58° C., 1 min 30 seconds at 68° C.; with a final reactionstep of 68° C. for 7 mins. The PCR products were evaluated by agarosegel electrophoresis, and desired bands were gel purified and sequencedusing an ABI 3730 DNA Analyzer (ABI).

51 candidate genes (listed in Table 4) were cloned from F401, Wt, M207and M544 lines, and sequenced for single nucleotide polymorphism (SNP)detection.

TABLE 4 Listing of Candidate Genes for Screening Quinolinate SynthaseA-1 Pathogenesis related protein 1 Allene oxide synthase Allene oxidecyclase ET861088.1 Methyl esterase FH733463.1 TGACG-sequence specifictranscription factor FH129193.1 Aquaporin-Transport FH297656.1 Universalstress protein Universal stress protein Tabacum FH077657.1Scarecrow-like protein sequence FH864888.1 EIN3-binding F-box FH029529.14,5 DOPA dioxygenase protein FI010668.1 Ethylene-responsive EB430189Carboxylesterase transcription factor DW001704 Glutathione S EB683763Bifunctional transferase inhibitor/lipid transfer protein/seed storage 2S albumin DW002318 Serine/threonine DW004086 Superoxide dismutaseprotein kinase DW001733 Lipid transfer protein DW001944 Proteinphosphatase DIR1 2 C DW002033 EB683763 Bifunctional inhibitor/ lipidtransfer protein/seed storage 2 S albumin DW002318 Serine/threonineDW002576 Glycosyl hydrolase of protein kinase unknown function DUF1680EB683279 EB683763 EB683951 FG141784 (FAD Oxidoreductase) BBLa-Tabacumsequences BBLb BBLc BBLd Pdr1 Pdr2 Pdr3 Pdr5a Pdr5b NtMATE1 NtMATE2NtMATE3 WRKY8 EIG-I24 WRKY3 WRKY9 EIG-E17 AJ748263.1 QPT2 quinolinatephosphoribosyltransferase AJ748262.1 QPT1

Example 6—SNP Detection and Characterization of the Mutants

Full length QS genes from the FC401 control and from the M207 and M544mutant plants were cloned using AP23 and QSR1 primers which weredesigned using N. benthamiana QS gene sequence. The primer sequences areshown in Table 5. The cloned QS genes were sequenced and analyzed formutations.

One SNP from M207 line and one SNP from M544 line were identified, andboth SNPs were found to be located in the quinolinate synthase (QS) gene(FIG. 4).

In mutant line M544, the mutation is located 1546 nucleotides from thestart codon. This G to A mutation results in an amino acid change fromvaline to isoleucine. In mutant line M207, the mutation is located 1460nucleotides from the start codon. This G to A mutation results in anamino acid change from cysteine to tyrosine.

TABLE 5 Quinolinate Synthase Gene Screening Primers Name Primer SequenceSEQ ID NO AP23 ATCTTTGCTTCCTCGACTCCA 21 AP24 AGTTAAGCGGAGCTTGATCGT 22AP23Seq1 ATGAGGTGCTGGCGTTGA 23 AP23Seq2 TTCAGCGTAGCTGCCTTG 24 AP23Seq3GGAGTTTATCGGATGTCG 25 AP23Seq4 TGTGCAGTAAGGAATGCATC 26 AP23Seq5GTTGTGAGCCGGTTCTGCAC 27 AP235pEND ATGGACGCCGCAAATTTAGTCATGA 28 AP29F1AGTGACTCGGAAATAACTAAAGGGTTTT 29 AP29R1 AAGTATTAGTCACAACATGCAGAGATGAG 30AP29F2 GAGCTCTTTCCATCTCTAGTAATCACA 31 AP29R2 ATGTAGGTATCAGGACCATACAACACT32 QSR1 GTTCGAGGTAGAACCTACTAG 33

Quinolinate Synthase A CDS-Fc401

(SEQ ID NO: 7) ATGGATGCCGCAAATTTAGTCATGAAATCTTCCTTGTTTTCGAAATCCCCATGTCCCCTTTTTAGTTCTAAACTCATTCCTAGAGCACCACCCTCTGTCTTTACTCTGCCTTCTACCTTTAGACCCCTCGTTAAATGCATACAAGCTTCATTCCCACCAAACCCTGATTCCAAAAAACCCTCAAACAATTCAACCTTTACGTGTTCAGCTGTGACTTCCTTCCCTTCTCAACAATCTCAGCCTCACGCGCCTTCCGATGCCAAGCTCCAACTCCTGATCTCTGAATTCCAGTCCCTCGTCGAACCAATGGACCGCGTGAAACGCCTCTTGCACTACTCCACACTCCTCCCTCCAATGGACGCGTCCTTCAAAACCCCTGAGAATCGCGTACCGGGTTGCACTACACAGGTATGGCTGAACGTGAGTTTCGATGAGGCTGAGAACAGGATGAAATTTTTGGCGGACAGTGACTCGGAAATAACTAAAGGGTTTTGCGCGTGTTTGGTTTCGCTGCTGGACGGGGCTACTCCCGATGAGGTGCTGGCGTTGAAAACGGAGGACTTGAATGCTTTGAATGTTGCGGGGTTGAACGGGAAAGGATCGGCATCTAGGGCGAATACGTGGCATAATGTGTTGGTCAGCATGCAGAAAAGGACAAGGGCCTTAGTTGCGGAGCGTGAAGGCAGGCCGCGCGGCGAGCTCTTTCCATCTCTAGTAATCACAGCTGATGGTATCCAACCCCAAGGCAGCTACGCTGAAGCCCAGGCAAGGTTCCTGTTTCCTGATGAATCAAGGGTCCAAAAACTTGCCAATTTGCTAAAGGAGAAGAAAATAGGAGTTGTTGCTCATTTCTACATGGACCCTGAGGTGCAAGGTGTTCTAACTGCAGCGCAGAAGCTTTGGCCCCATATACATATATCTGATTCTTTAGTCATGGCTGATAAAGCTGTCAGTATGGCAAAAGCTGGATGTGAATATATATCTGTATTGGGTGTAGATTTCATGTCAGAGAATGTGCGAGCCATTCTTGATCTAGCTGGATTCCCAGAGGTTGGAGTTTATCGGATGTCGGACGAACGCATTGGTTGTTCTTTGGCTGATGCTGCAGCCAGCCCAGCATACTTGGATTATCTTAAAACAGCTTCAACTTCTTCTCCATCTCTGCATGTTGTGTACATAAATACTTCACTGGAGACAAAAGCATATTCTCATGAGCTTGTTCCGACTATAACATGTACTTCCTCTAATGTTGTGCAAACTATTCTGCAGGCATTTGCTGAAGTACCTGACTTGGAAGTGTTGTATGGTCCTGATACCTACATGGGTTCAAACATTGCGGAATTGTTCACCCAGATGTCCACGATGACTGATGAAGAAATTTCTGCGATACATCCTTTGCACAACAGAATCTCCATTAAATCTTTGCTTCCTCGACTGCATTATTTTCAGGATGGGACATGTATTGTTCATCACCTCTTTGGTCATGAAGTTGTGGAGAAGATAAATGAAATGTATGGGGATGCATTCCTTACTGCACACTTTGAAGTTCCTGGTGAAATGTTTTCCCTGGCAATGGAAGCGAAGAAAAGGGGCATGGGAGTAGTAGGTTCTACCTCGAACATACTCGACTTTATCAAAGAAAGGGTAGAAGAGTCCTTGAATAGAAACGTAGATGAACATCTTCAGTTTGTTTTGGGAACGGAATCAGGAATGATTACGGCAATAGTTGCAGCAGTCGGTAAATTACTAGGTTCTGCTGACTCCTCTTCCGGTGGAGCAAAAGTAAGTGTTGAGATTGTCTTTCCTGTCTCGTCAGAATCAGTGACAAGAACATCTACGGGTTCGCCTCTGGACCAAAATAAGGTCAATATTATACCTGGAGTTGCAAGTGGAGAGGGGTGTTCTCTACATGGTGGATGTGCCTCCTGTCCATATATGAAGATGAACTCTCTTAGCTCGTTGCTAAAAGTTTGCCAGAGCTTGCCCCATGGCAAAGCCGAACTTTCAGCTTATGAGGCAGGACGATTCAGTTTGCGAACCCCCAAGGGAAAACAAATTGCGGATGTTGGTTGTGAGCCGGTTCTGCACATGAGACACTTTCAGGCAACAAAGAGATTACCAGAGCAGCTAATCAATCAAATACTTCAACG ATCAAGCTCTGCTTAA(SEQ ID NO: 8) MDAANLVMKSSLFSKSPCPLFSSKLIPRAPPSVFTLPSTFRPLVKCIQASFPPNPDSKKPSNNSTFTCSAVTSFPSQQSQPHAPSDAKLQLLISEFQSLVEPMDRVKRLLHYSTLLPPMDASFKTPENRVPGCTTQVWLNVSFDEAENRMKFLADSDSEITKGFCACLVSLLDGATPDEVLALKTEDLNALNVAGLNGKGSASRANTWHNVLVSMQKRTRALVAEREGRPRGELFPSLVITADGIQPQGSYAEAQARFLFPDESRVQKLANLLKEKKIGVVAHFYMDPEVQGVLTAAQKLWPHIHISDSLVMADKAVSMAKAGCEYISVLGVDFMSENVRAILDLAGFPEVGVYRMSDERIGCSLADAAASPAYLDYLKTASTSSPSLHVVYINTSLETKAYSHELVPTITCTSSNVVQTILQAFAEVPDLEVLYGPDTYMGSNIAELFTQMSTMTDEEISAIHPLHNRISIKSLLPRLHYFQDGTCIVHHLFGHEVVEKINEMYGDAFLTAHFEVPGEMFSLAMEAKKRGMGVVGSTSNILDFIKERVEESLNRNVDEHLQFVLGTESGMITAIVAAVGKLLGSADSSSGGAKVSVEIVFPVSSESVTRTSTGSPLDQNKVNIIPGVASGEGCSLHGGCASCPYMKMNSLSSLLKVCQSLPHGKAELSAYEAGRFSLRTPKGKQIADVGCEPVLHMRHF QATKRLPEQLINQILQRSSSA

There are 2787 QS genes listed in Pfam protein data base and 41 of theQS genes are from plants (The Pfam protein family database; Punta etal., Nuc. Acids Res., 2012, 40:D290-D301). The alignment that is shownin FIG. 5 compares the amino acid sequences of the wild type QS and thetwo mutant lines with respect to representative plant QS sequences. TheM207 mutation (C to Y) is conserved in all but one of the 2787 QSsequences deposited in the Pfam database. The M544 mutation (V to I) isconserved only in plants and not in other QS genes.

To confirm the stability of the mutations, the QS gene was sequenced inmultiple generations of mutants. The QS gene mutation patternscorrelated with the anatabine levels of the mutant lines, indicatingthat the QS gene mutation is responsible for the ultra-low anatabinetrait. See Table 6.

TABLE 6 Stability of mutant lines Line ID M2 Seed M3 Seed M4 Seed SNPM207 MS103 MS4144 MS13379 Homozygous Homozygous Hetero- Homo- MS13396Homozygous Homozygous zygous zygous MS13397 Homozygous HomozygousMS13398 Homozygous Homozygous MS13399 Homozygous Homozygous MS13400Heterozygous Heterozygous MS13401 Homozygous Homozygous MS13473Homozygous Homozygous MS13537 Homozygous Homozygous MS13374 HomozygousHomozygous MS12469 MS13391 Homozygous Homozygous Hetero- MS13392Homozygous Homozygous zygous MS13393 Homozygous Homozygous M544 MS445M54360 MS13474 Homozygous Homozygous Hetero- Homo- MS13540 HomozygousHomozygous zygous zygous MS12471 MS13370 Homozygous Homozygous Hetero-MS13371 Homozygous Homozygous zygous MS13389 Homozygous Homozygous

Example 7—Confirmation of Linkage Between the Quinolinate Synthase Geneand the Ultra-Low Anatabine Phenotype

In order to verify the linkage between the low anatabine phenotype andthe QS SNP genotype, a mapping population was tested for both anatabinelevel and SNPs. The mapping population was generated previously bycrossing mutant line M207 and wild type Red Russian. FIG. 6 shows thelinkage between the anatabine trait, the PT60808 genetic marker, whichis the genetic marker that is the most tightly linked to the trait, andthe QS mutation. From 115 F2 plants tested, the QS mutationco-segregated with the low anatabine trait.

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

1. A tobacco plant having an induced mutation in an endogenous gene,wherein the endogenous gene has a sequence at least 90% identical to SEQID NO:
 7. 2. The tobacco plant of claim 1, wherein the mutant plantexhibits reduced expression or activity of quinolinate synthase ascompared to a control plant lacking the induced mutation.
 3. The tobaccoplant of claim 1, wherein leaf from the mutant plant comprises a reducedamount of at least one alkaloid as compared to leaf from a control plantlacking the induced mutation.
 4. The tobacco plant of claim 3, whereinthe alkaloid is anatabine.
 5. The tobacco plant of claim 1, whereincured leaf from the mutant plant exhibits a reduced amount of at leastone tobacco-specific nitrosamine (TSNA) as compared to cured leaf from acontrol tobacco plant lacking the induced mutation.
 6. The tobacco plantof claim 5, wherein the at least one TSNA is N′-nitrosoanatabine (NAT).7. Seed produced by the tobacco plant of claim 1, wherein the seedcomprises the induced mutation. 8-31. (canceled)
 32. The tobacco plantof claim 1, wherein the induced mutation provides an amino acidsubstitution at a position corresponding to the cysteine residue atposition 487 or the valine residue at position 516 of SEQ ID NO:
 8. 33.The tobacco plant of claim 1, wherein the induced mutation comprises atyrosine at amino acid position 487 of SEQ ID NO:
 8. 34. The tobaccoplant of claim 1, wherein the induced mutation comprises an isoleucineat position 516 of SEQ ID NO:
 516. 35. The tobacco plant of claim 4,wherein the reduced amount of anatabine comprises a reduction of about50% to about 95% as compared to a control plant lacking the inducedmutation.
 36. The tobacco plant of claim 1, wherein cured leaf from thetobacco plant comprises an anatabine percentage of less than 0.5%. 37.The tobacco plant of claim 1, wherein the endogenous gene is at least95% identical to SEQ ID NO:
 7. 38. The tobacco plant of claim 1, whereinthe endogenous gene is 100% identical to SEQ ID NO:
 7. 39. The tobaccoplant of claim 1, wherein the induced mutation is in a residue fullyconserved in the wild-type quinolinate synthase proteins shown in FIG.5.
 40. A method for producing a tobacco seed comprising: (a) crossing afirst tobacco plant having an induced mutation in an endogenous gene,wherein the endogenous gene has a sequence at least 90% identical to SEQID NO: 7 with a second tobacco plant to produce at least one progenyseed; and (b) selecting at least one progeny seed from step (a) thatcomprises said induced mutation.
 41. The method of claim 40, wherein thefirst tobacco plant exhibits reduced expression or activity ofquinolinate synthase as compared to a control plant lacking the inducedmutation.
 42. The method of claim 40, wherein the induced mutationprovides an amino acid substitution at a position corresponding to thecysteine residue at position 487 or the valine residue at position 516of SEQ ID NO:
 8. 43. A method of making a tobacco plant comprising: (a)inducing mutagenesis in a Nicotiana tabacum cell to produce amutagenized cell; (b) obtaining one or more plants regenerated from themutagenized cell of step (a); and (c) identifying at least one plantfrom step (b) that comprises an induced mutation in an endogenous gene,wherein the endogenous gene has a sequence at least 90% identical to SEQID NO:
 7. 44. The method of claim 43, wherein the at least one plantidentified in step (c) exhibits reduced expression or activity ofquinolinate synthase as compared to a control plant lacking the inducedmutation.